Construction and detection of expression vectors of microRNA-9a in BmN cells  

作　　者：Yong HUANG Quan ZOU Sheng-peng WANG Shun-ming TANG Guo-zheng ZHANG Xing-jia SHEN 

机构地区：[1]Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Jiangsu University of Science and Technology, Zhenjiang 212018, China [2]School of lnformation Science and Technology, Xiamen University, Xiamen 361005, China [3]Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China [4]Animal Science and Technology College, Henan University of Science and Technology, Luoyang 471003, China

出　　处：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2011年第7期527-533,共7页浙江大学学报（英文版）B辑（生物医学与生物技术）

基　　金：Project supported by the National Basic Research Program (973) of China (No. 2005CB121004);the National High-Tech R & D Program (863) of China (No. 2006AA10A119);the Innovation Foundation for Graduate Students of Jiangsu Province;the National Natural Science Foundation of China (No. 61001013)

摘　　要：MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.MicroRNAs （miRNAs） are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions （UTRs） of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 （A3） promoter and flanked sequences of miRNA-9a （miR-9a） precursor were amplified from genomic DNA of the silkworm （Bombyx mori） and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein （EGFP） gene was used as reporter gene.The Bombyx mori N （BmN） cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR） and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

关 键 词：miRNA-9a （miR-9a） EGFP gene Bombyx mori N （BmN） Cells Expression vector 

分 类 号：Q34[生物学—遗传学]